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  • br biomarkers for CRC diagnostic strategies particularly


    biomarkers for CRC diagnostic strategies, particularly in the early stage, is urgently needed in the improvement of clinical outcomes.
    Long noncoding RNAs (lncRNAs) are commonly > 200 nucleotides (nt) in length with limited potential in protein coding [5]. Emerging studies have demonstrated that lncRNAs play important role in cancer biology, carcinogenesis and metastasis and have been regarded as po-tential diagnostic and prognostic factors [6–8]. More importantly, it is feasible to detect cancer-related lncRNAs in blood and other body fluids [9–11], which shows the reliable potentiality as novel tumor marker. Colon cancer-associated transcript 2 (CCAT2), which maps to the 8q24 region and encompasses the rs6983267 single nucleotide poly-morphism, has been proved to play a crucial role in different cancers including colon, gastric, breast and hepatocellular carcinoma [12–16]. Ling et al. found that CCAT2 was highly over-expressed in
    Abbreviations: CRC, colorectal cancer; LncRNAs, long noncoding RNAs; CCAT2, colon cancer-associated transcript 2; UICC, International Cancer Control; TNM, tumor-node-metastasis; RT-qPCR, quantitative real-time PCR; NTA, nanoparticle tracking analysis; ANOVA, one-way analysis of variance; NC, Negative control
    Corresponding author at: Department of Clinical Laboratry, The second Hospital of Shandong University, 247 Beiyuan Street, Jinan, China. E-mail address: [email protected] (C. Wang).
    Fig. 1. High CCAT2 G-418 is associated with the progression of tumor development in CRC patients.
    Notes: (A) CCAT2 expression was determined by qRT-PCR and normalized against GAPDH in 75 paired CRC tissues and adjacent non-cancerous tissues. (B) The level of CCAT2 in TNM stages of CRC. (C) The over-expression of CCAT2 in CRC was associated with lymph node metastasis. (D) The increased levels of CCAT2 were associated with deeper invasion in CRC.
    microsatellite-stable colorectal cancer in Italian, Japanese, Australian and Northern American cohort. In addition, it’s revealed that CCAT2 was up-regulated in CRC cell lines and involved in tumor growth and metastasis [17]. Recently, Zhang et al. showed that CCAT2 was oever-expressed in CRC tissues and progressively increased with tumor stages [18]. However, to the best of our knowledge, the clinical significance of circulating CCAT2 expression in CRC has not previously been eluci-dated.
    In the present study, we investigated the level of CCAT2 expression in colorectal cancer tissues and adjacent non-cancerous tissues, fol-lowed with the analysis of serum CCAT2 expression in CRC patients and healthy controls. Furthermore, we investigated the existence of serum CCAT2 in the exosomes, which support that exosomal-sourced CCAT2 may be a novel predictor for CRC.
    2. Materials and methods
    2.1. Patients and control subjects
    All patients and control subjects involved in this study were re-cruited from Qilu Hospital, Shandong University. A total of 75 resected tumor tissues and adjacent non-cancerous tissues (at least 5 cm away from tumor margin) were obtained in the Department of General 
    Surgery. The diagnosis of CRC was histopathologically confirmed and other clinical data including age, gender, tumor location, tumor size, differentiation, lymph node metastasis and distant metastasis were collected. The postoperative pathological staging was evaluated based on the 7th edition of the Union for International Cancer Control (UICC) tumor-node-metastasis (TNM) staging system. Control samples were obtained from healthy volunteers. This study was approved by the Ethics Committee of the Qilu Hospital of Shandong University.
    2.2. Sample collection and preparation
    All resected tissue samples were snap-frozen in liquid nitrogen im-mediately, followed by storing at -80℃. Venous blood was collected and prepared as previously described and stored at -80℃ until analysis.
    2.3. Isolation of exosomes
    A total of 5 serum samples were randomly divided into two equal parts. One part was used to extract total RNA using TRIzol and TRIzol LS reagents, and the other was used to isolate exosomes using ExoQuick solution (System Biosciences, Mountain View, CA). Briefly, the filtered serum was added by ExoQuick solution and incubated for 30 min, fol-lowed by centrifugation at 1500 g for 30 min. The pellets of exosomes
    Table 1
    The relationship between CCAT2 expression and demographic characteristics of patients with CRC.
    Characteristic Tissue sample of CRC patients
    Serum sample of CRC patients
    Age (year)
    Tumor location
    Local invasion
    Lymph node
    TNM stages
    TNM, Tumor node metastasis; *, statistical significance.
    Fig. 2. The expression levels of Serum CCAT2 in CRC patients.
    Notes: The expression levels of serum lncRNA CCAT2 was significantly higher in the CRC group compared with the control group.